A non-capacitative pathway activated by arachidonic acid is the major Ca2+ entry mechanism in rat A7r5 smooth muscle cells stimulated with low concentrations of vasopressin

Depletion of the Ca 2+ stores of A7r5 cells stimulated Ca 2+ , though not Sr 2+ , entry. Vasopressin (AVP) or platelet-derived growth factor (PDGF) stimulated Sr 2+ entry. The cells therefore express a capacitative pathway activated by empty stores and a non-capacitative pathway stimulated by receptors; only the former is permeable to Mn 2+ and only the latter to Sr 2+ . Neither empty stores nor inositol 1,4,5-trisphosphate (Ins P 3 ) binding to its receptors are required for activation of the non-capacitative pathway, because microinjection of cells with heparin prevented PDGF-evoked Ca 2+ mobilization but not Sr 2+ entry. Low concentrations of Gd 3 + irreversibly blocked capacitative Ca 2+ entry without affecting AVP-evoked Sr 2+ entry. After inhibition of the capacitative pathway with Gd 3 + , AVP evoked a substantial increase in cytosolic [Ca 2+ ], confirming that the non-capacitative pathway can evoke a significant increase in cytosolic [Ca 2+ ]. Arachidonic acid mimicked the effect of AVP on Sr 2+ entry without stimulating Mn 2+ entry; the Sr 2+ entry was inhibited by 100 μM Gd 3 + , but not by 1 μM Gd 3 + which completely inhibited capacitative Ca 2+ entry. The effects of arachidonic acid did not require its metabolism. AVP-evoked Sr 2+ entry was unaffected by isotetrandrine, an inhibitor of G protein-coupled phospholipase A 2 . U73122, an inhibitor of phosphoinositidase C, inhibited AVP-evoked formation of inositol phosphates and Sr 2+ entry. The effects of phorbol esters and Ro31-8220 (a protein kinase C inhibitor) established that protein kinase C did not mediate the effects of AVP on the non-capacitative pathway. An inhibitor of diacylglycerol lipase, RHC-80267, inhibited AVP-evoked Sr 2+ entry without affecting capacitative Ca 2+ entry or release of Ca 2+ stores. Selective inhibition of capacitative Ca 2+ entry with Gd 3 + revealed that the non-capacitative pathway is the major route for the Ca 2+ entry evoked by low AVP concentrations. We conclude that in A7r5 cells, the Ca 2+ entry evoked by low concentrations of AVP is mediated largely by a non-capacitative pathway directly regulated by arachidonic acid produced by the sequential activities of phosphoinositidase C and diacylglycerol lipase.